This invention relates to apoptosis, tumor necrosis factor-xcex1 (TNF-xcex1) mediated signalling, cell cycle and tumor growth suppression.
Apoptosis is a morphologically distinct form of programmed cell death that is important in the normal development and maintenance of multicellular organisms. Dysregulation of apoptosis can take the form of inappropriate suppression of cell death, as occurs in the development of cancers, or in a failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders.
Some baculoviruses encode proteins termed xe2x80x9cinhibitors of apoptosis proteinsxe2x80x9d (IAPs) because they inhibit the apoptosis that would otherwise occur when insect cells are infected by the virus. These proteins are thought to work in a manner that is independent of other viral proteins. The baculovirus IAP genes include sequences encoding a ring zinc finger-like motif (RZF), which may be involved in DNA binding, and two N-terminal domains that consist of a 70 amino acid repeat motif termed a BIR domain (Baculovirus IAP Repeat).
We have recently discovered a mammalian family of IAP polypeptides. These polypeptides include the human proteins HIAP-1, HIAP-2, and XIAP and their murine homologs. A related protein, NAIP, has also been found. The mammalian IAP levels have been shown to be increased both in cancer cells and cells which survive events known to induce apoptosis (e.g., ischemia). The IAPs have also been shown to block apoptosis triggered by diverse stimuli. These results are consistent with a role for the mammalian IAPs as inhibitors of apoptosis.
The IAP family is now known to include at least two Drosophila proteins, in addition to the original four mammalian homologues (Hay et al., Cell 83: 1253-1262, 1995). Although we and others have established that the IAPs can suppress apoptosis in tissue culture model systems their mechanism of action is still under investigation.
We have discovered a novel family of genes, the XAFs. Members of the XAF gene family encode proteins that interact with IAPs and are associated with apoptosis. Our discovery allows the development of diagnostic, prognostic, and therapeutic compounds and methods for the detection and treatment of diseases involving apoptosis.
In a first aspect, the invention features substantially pure nucleic acid encoding a XAF polypeptide.
In a second aspect, the invention features substantially pure nucleic acid corresponding to at least ten nucleotides of a nucleic acid encoding a XAF polypeptide, where the nucleic acid is antisense nucleic acid and the antisense nucleic acid is sufficient to decrease XAF biological activity. In various embodiments of this aspect, the antisense nucleic acid corresponds to at least fifteen nucleotides of a nucleic acid encoding a XAF polypeptide, at least thirty nucleotides of a nucleic acid encoding a XAF polypeptide, or at least 100 nucleotides of a nucleic acid encoding a XAF polypeptide. In other embodiments, the XAF biological activity is decreased by at least 20%, 40%, 60%, or 80%. In yet another embodiment of this aspect of the invention, the antisense nucleic acid is in a vector where the vector is capable of directing expression of the antisense nucleic acid in a vector-containing cell.
In a third aspect, the invention features a vector that includes a substantially pure nucleic acid encoding a XAF polypeptide, where the vector is capable of directing expression of the polypeptide in a vector-containing cell.
In another related aspect, the invention features a cell that contains a substantially pure nucleic acid encoding a XAF polypeptide. In a preferred embodiment of this aspect, the nucleic acid is expressed in the cell. In various preferred embodiments, the cell is present in a patient having a disease that is caused by excessive or insufficient cell death and the cell is selected from the group that includes a fibroblast, a neuron, a glial cell, an insect cell, an embryonic stem cell, a myocardial cell, and a lymphocyte.
In a fifth aspect, the invention features a transgenic animal generated from a cell genetically engineered to lack nucleic acid encoding a XAF polypeptide, where the transgenic animal lacks expression of the XAF polypeptide.
In a related aspect, the invention features a transgenic animal generated from a cell that contains a substantially pure nucleic acid that replaces DNA encoding a XAF polypeptide, where the nucleic acid is expressed in the transgenic animal.
In various embodiments of this aspect, the XAF polypeptide is from a mammal (e.g., a human or a rodent). In another embodiment, the nucleic acid is genomic DNA or cDNA, and is operably linked to regulatory sequences for expression of the polypeptide where the regulatory sequences include a promoter (e.g., a constitutive promoter, a promoter inducible by one or more external agents, or a cell-type specific promoter). In other preferred embodiments, the XAF polypeptide is selected from a group that includes XAF-1, XAF-2 N terminus, XAF-2L and XAF-2S. In another embodiment, the XAF-1 has the amino acid sequence of SEQ ID NO.: 2 or the nucleic acid sequence of SEQ ID NO.: 1, and may include a deletion of the nucleic acids encoding the carboxy terminal amino acids 173 to 317 of XAF-1 (SEQ ID NO.: 8); or a deletion of the nucleic acids encoding the amino terminal amino acids 1 to 172 of XAF-1 (SEQ ID NO.: 7). In another embodiment of this aspect of the invention, the XAF-2 N terminus polypeptide has the amino acid sequence of SEQ ID NO.: 4 or the nucleic acid sequence of SEQ ID NO.: 3. In another embodiment, the XAF-2L polypeptide has the amino acid sequence of SEQ ID NO.: 10 or the nucleic acid sequence of SEQ ID NO.: 9. In yet another embodiment, the XAF-2S polypeptide has the amino acid sequence of SEQ ID NO.: 12 or the nucleic acid sequence of SEQ ID NO.: 11.
In a seventh aspect, the invention features a method of identifying a compound that modulates apoptosis. The method includes: (a) providing a cell that has a XAF gene; (b) contacting the cell with a candidate compound; and (c) monitoring expression of the XAF gene, where an alteration in the level of expression of the XAF gene indicates the presence of a compound which modulates apoptosis. In one preferred embodiment of this aspect, the alteration that is an increase indicates the compound is increasing apoptosis, and the alteration that is a decrease indicates the compound is decreasing apoptosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
In a related aspect, the invention features another method of identifying a compound that is able to modulate apoptosis that includes: (a) providing a cell including a reporter gene operably linked to a promoter from a XAF gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where a change in the expression in response to the candidate compound identifies a compound that is able to modulate apoptosis. In one preferred embodiment of this aspect, the alteration that is an increase indicates the compound is increasing apoptosis, and the alteration that is a decrease indicates the compound is decreasing apoptosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
In a ninth aspect, the invention features a method of identifying a compound that is able to inhibit XAF-mediated apoptosis that includes: (a) providing a cell expressing an apoptosis-inducing amount of XAF; (b) contacting the cell with a candidate compound; and (c) measuring the level of apoptosis in the cell, where a decrease in the level relative to a level in a cell not contacted with the candidate compound indicates a compound that able to inhibit XAF-mediated apoptosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
In a tenth aspect, the invention features a method of identifying a compound that is able to induce XAF-mediated apoptosis that includes: (a) providing a cell expressing an apoptosis-inducing amount of XAF; (b) contacting the cell with a candidate compound; and (c) measuring level of apoptosis in the cell, where an increase in the level relative to a level in a cell not contacted with the candidate compound indicates a compound that able to induce XAF-mediated apoptosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
In related aspects, the invention features other methods of identifying a compound that is able to modulate apoptosis
One such method includes: (a) providing a cell expressing a TRAF polypeptide, a XAF polypeptide, and a reporter gene operably linked to DNA that includes an NF-xcexaB binding site; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where a change in expression in response to the compound indicates that the compound is able to modulate apoptosis. In a preferred embodiment of this aspect of the invention, the TRAF is selected from a group that includes TRAF2, TRAF5, and TRAF6. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
A second such method includes: (a) providing a cell expressing a TRAF polypeptide, a XAF polypeptide, an IAP polypeptide, and a reporter gene operably linked to DNA that includes an NF-xcexaB binding site; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where a change in expression in response to the compound indicates that the compound is able to modulate apoptosis. In a preferred embodiment of this aspect of the invention, the IAP is XIAP. In another preferred embodiment of this aspect of the invention, the TRAF is selected from a group that includes TRAF2, TRAF5, and TRAF6. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
A third such method includes: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein, where the first fusion protein includes a XAF polypeptide covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site; (iii) a second fusion gene capable of expressing a second fusion protein, where the second fusion protein includes a XAF polypeptide covalently bonded to a gene activating moiety; (b) exposing the cell to the compound; and (c) measuring reporter gene expression in the cell, where a change in the reporter gene expression indicates that the compound is capable of modulating apoptosis. In a preferred embodiment of this aspect of the invention, the cell is a yeast cell.
A fourth method for detecting a compound capable of modulating apoptosis includes: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein, where the first fusion protein includes a XAF polypeptide covalently bonded to a binding moiety capable of specifically binding to the DNA-bindingprotein recognition site; (iii) a second fusion gene capable of expressing a second fusion protein, where the second fusion protein includes an IAP polypeptide covalently bonded to a gene activating moiety; (b) exposing the cell to the compound; and (c) measuring reporter gene expression in the cell, where a change in the reporter gene expression indicates that the compound is capable of modulating apoptosis. In a preferred embodiment of this aspect of the invention, the IAP is XIAP. In another preferred embodiment, the cell is a yeast cell.
A fifth such method includes: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein, where the first fusion protein includes an IAP polypeptide covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site; (iii) a second fusion gene capable of expressing a second fusion protein, where the second fusion protein includes a XAF polypeptide covalently bonded to a gene activating moiety; (b) exposing the cell to the compound; and (c) measuring reporter gene expression in the cell, where a change in the reporter gene expression indicates that the compound is capable of modulating apoptosis. In a preferred embodiment of this aspect of the invention, the IAP is XIAP. In another preferred embodiment, the cell is a yeast cell.
A sixth such method includes: (a) providing a first XAF polypeptide immobilized on a solid-phase substrate; (b) contacting the first XAF polypeptide with a second XAF polypeptide; (c) contacting the first XAF polypeptide and the second XAF polypeptide with a compound; and (d) measuring amount of binding of the first XAF polypeptide to the second XAF polypeptide, where a change in the amount relative to an amount not contacted with the compound indicates that the compound is capable of modulating apoptosis.
A seventh method for detecting a compound capable of modulating apoptosis includes: (a) contacting a XAF polypeptide immobilized on a solid-phase substrate; (b) providing the XAF polypeptide with an IAP polypeptide; (c) contacting the XAF polypeptide and the IAP polypeptide with a compound; and (d) measuring amount of binding of the XAF polypeptide to the IAP polypeptide, where a change in the amount relative to an amount not contacted with the compound indicates that the compound is capable of modulating apoptosis. In a preferred embodiment of this aspect of the invention, the IAP is XIAP.
An eighth such method includes: (a) providing an IAP polypeptide immobilized on a solid-phase substrate; (b) contacting the IAP polypeptide with a XAF polypeptide; (c) contacting the IAP polypeptide and the XAF polypeptide with a compound; and (d) measuring amount of binding of the IAP polypeptide to the XAF polypeptide, where a change in the amount relative to an amount not contacted with the compound indicates that the compound is capable of modulating apoptosis. In a preferred embodiment of this aspect of the invention, the IAP is XIAP.
In various preferred embodiments of the seventh to eighteenth method aspects of the invention, the XAF is XAF-1; the XAF is the N-terminus of XAF-2; the XAF is XAF-2L, or the XAF is XAF-2S. In other embodiments, the XAF is from a mammal (e.g., a human or a rodent).
In a nineteenth aspect, the invention features a method of increasing apoptosis in a cell by administering to the cell an apoptosis inducing amount of XAF polypeptide or fragment thereof.
In related aspects, the invention includes methods of increasing apoptosis by either providing a transgene encoding a XAF polypeptide or fragment thereof to a cell of an animal such that the transgene is positioned for expression in the cell; or by administering to the cell a compound which increases XAF biological activity in a cell (e.g., by administering a polypeptide fragment of a XAF polypeptide, a mutant of a XAF polypeptide, or a nucleic acid encoding a XAF polypeptide, a mutant thereof, or a polypeptide fragment thereof).
In preferred embodiment of the nineteenth, twentieth, and twenty-first aspects of the invention, the XAF is selected from a group that includes XAF-1, XAF-2 N-terminus, XAF-2L, and XAF-2S. In various preferred embodiments, the XAF is from a mammal (e.g., a human or rodent); the cell is in a mammal (e.g., a human or rodent); the cell is in an mammal diagnosed as having a condition involving insufficient apoptosis, (e.g., a cancer such as breast cancer, uterine cervical carcinoma, gastric carcinoma, ovarian epithelial cancer, pediatric medulloblastoma, lung carcinoma, prostate cancer); and the cell is a peripheral blood leukocyte (e.g., a lymphocyte), a muscle cell (e.g., a myocardial cell), an intestinal cell, an ovarian cell, a placental cell, or a thymus cell (e.g., a thymocyte).
In a twenty-second aspect, the invention features a method of inhibiting apoptosis in a cell, by administering to the cell an apoptosis-inhibiting amount of XAF polypeptide or fragment thereof.
In related aspects, the invention features a method of inhibiting apoptosis in a cell by providing to the cell a transgene encoding a XAF polypeptide or fragment positioned for expression in the cell; and a method of inhibiting apoptosis by administering a compound which decreases XAF biological activity (e.g., an antibody which specifically binds to a XAF polypeptide (e.g., a neutralizing antibody), a polypeptide fragment of a XAF polypeptide, a mutant form of a XAF polypeptide, an antisense nucleic acid complementary to the XAF coding sequence, a negative regulator of the XAF-dependent apoptotic pathway, or a XAF antisense nucleic acid).
In a preferred embodiment of the twenty-second, twenty-third, and twenty-fourth aspects of the invention, the XAF is selected from a group that includes XAF-1, XAF-2 N-terminus, XAF-2L, and XAF-2S. In various preferred embodiments, the XAF is from a mammal (e.g., a human or rodent); the cell is in a mammal (e.g., a human or rodent); and the mammal bearing the cell is an mammal diagnosed as having a condition involving excessive apoptosis (e.g., AIDS, a neurodegenerative disease, a myelodysplastic syndrome, or an ischemic injury (caused by, e.g., a myocardial infarction, a stroke, or a reperfusion injury, a toxin-induced liver disease, physical injury, renal failure, a secondary exsaunguination or blood flow interruption resulting from any other primary diseases)). In other preferred embodiments, the cell is a muscle cell (e.g., a myocardial cell), a peripheral blood leukocyte (e.g., a lymphocyte, such as a T lymphocyte (preferably, a CD4+ T lymphocyte)), an intestinal cell, an ovarian cell, a placental cell, a thymus cell (e.g., a thymocyte), or a breast cell.
In the twenty-fifth and twenty-sixth aspects, the invention features methods of diagnosing a mammal for the presence of disease involving altered apoptosis or an increased likelihood of developing a disease involving altered apoptosis. The methods include isolating a sample of nucleic acid from the mammal and determining whether the nucleic acid includes a XAF mutation, where the presence of a mutation is an indication that the animal has an apoptosis disease or an increased likelihood of developing a disease involving apoptosis; or measuring XAF gene expression in a sample from an animal to be diagnosed, where an alteration in the expression or activity relative to a sample from an unaffected mammal is an indication that the mammal has a disease involving apoptosis or increased likelihood of developing such a disease. In preferred embodiments, XAF gene expression is measured by assaying the amount of XAF polypeptide or XAF biological activity in the sample (e.g., the XAF polypeptide is measured by immunological methods), or XAF gene expression is measured by assaying the amount of XAF RNA in the sample.
In one preferred embodiment of the twenty-fifth and twenty-sixth of the invention, the XAF is selected from a group that includes XAF-1, XAF-2 N-terminus, XAF-2L, and XAF-2S. In another preferred embodiment, the mammal is a human.
In a twenty-seventh aspect, the invention features a kit for diagnosing a mammal for the presence of a disease involving altered apoptosis or an increased likelihood of developing a disease involving altered apoptosis that includes a substantially pure antibody that specifically binds a XAF polypeptide.
Another such kit includes a material for measuring XAF RNA (e.g., a probe). in a preferred embodiment, the material is a nucleic acid probe.
A third such kit includes both a substantially pure antibody that specifically binds a XAF polypeptide, as well as a material for measuring XAF RNA. In a preferred embodiment, the kit also includes a means for detecting the binding of the antibody to the XAF polypeptide. In another preferred embodiment, the material is a nucleic acid probe.
In a thirtieth aspect, the invention features a method of obtaining a XAF polypeptide, including: (a) providing a cell with DNA encoding a XAF polypeptide, the DNA being positioned for expression in the cell; (b) culturing the cell under conditions for expressing the DNA; and (c) isolating the XAF polypeptide.
In preferred embodiments of this aspect of the invention, the XAF is XAF-1, XAF-2 N terminus, XAF-2L, or XAF-2S. In another preferred embodiment, the DNA further includes a promoter inducible by one or more external agents.
In a thirty-first aspect, the invention features a method of isolating a XAF gene or portion thereof having sequence identity to human XAF-1. The method includes amplifying by polymerase chain reaction the XAF gene or portion thereof using oligonucleotide primers wherein the primers (a) are each greater than 13 nucleotides in length; (b) each have regions of complementarity to opposite DNA strands in a region of the nucleotide sequence of FIG. 1; and (c) optionally contain sequences capable of producing restriction endonuclease cut sites in the amplified product; and isolating the XAF gene or portion thereof.
In a related aspect, the invention features a method of isolating a XAF gene or portion thereof having sequence identity to human XAF-2L or XAF-2S. The method includes amplifying by polymerase chain reaction the XAF gene or portion thereof using oligonucleotide primers wherein the primers (a) are each greater than 13 nucleotides in length; (b) each have regions of complementarity to opposite DNA strands in a region of the nucleotide sequence of FIG. 37A; and (c) optionally contain sequences capable of producing restriction endonuclease cut sites in the amplified product; and isolating the XAF gene or portion thereof.
In another related aspect, the invention features a method of isolating a XAF gene or fragment thereof from a cell, including the steps of: (a) providing a sample of cellular DNA; (b) providing a pair of oligonucleotides having sequence homology to a conserved region of a XAF gene; (c) combining the pair of oligonucleotides with the cellular DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and (d) isolating the amplified XAF gene or fragment thereof. In a preferred embodiment of the above three aspects, the polymerase chain reaction is reverse-transcription polymerase chain reaction (e.g., RACE).
In yet another related aspect, the invention features a method of identifying a XAF gene in a mammalian cell that includes: (a) providing a preparation of mammalian cellular DNA; (b) providing a detectably-labeled DNA sequence having identity to a conserved region of a second known XAF gene; and (c) contacting the preparation of cellular DNA with the detectably-labeled DNA sequence under hybridization conditions that provide detection of a gene having 50% or greater nucleotide sequence identity to the detectably-labeled DNA sequence; and identifying the XAF gene. In one preferred embodiment of this method for detecting a XAF gene, the DNA sequence includes at least a portion of XAF-1. In another preferred embodiment, the DNA sequence includes at least a portion of XAF-2L. In another preferred embodiment, the DNA sequence includes at least a portion of XAF-2S.
In a thirty-fifth aspect, the invention features a method for identifying a XAF gene that includes the steps of: (a) providing a mammalian cell sample; (b) introducing by transformation into the cell sample a candidate XAF gene; (c) expressing the candidate XAF gene within the cell sample; and (d) determining whether the sample exhibits an altered level of apoptosis, where an alteration in the level of apoptosis identifies a XAF gene. Preferably, the alteration is an increase in apoptosis and the cell is a leukocyte, a fibroblast, an insect cell, a glial cell, a myocardial cell, an embryonic stem cell, or a neuron.
In other aspects, the invention features a XAF nucleic acid for use in modulating apoptosis, a XAF polypeptide for use in modulating apoptosis, the use of a XAF polypeptide for the manufacture of a medicament for the modulation of apoptosis, and the use of a XAF nucleic acid for the manufacture of a medicament for the modulation of apoptosis. Preferably, the XAF is selected from a group that includes XAF-1, XAF-2 N terminus, XAF-2L, and XAF-2S.
In a fortieth aspect, the invention features a substantially pure antibody that specifically binds a XAF polypeptide, or a fragment or a mutant thereof. In one preferred embodiment of this aspect, the XAF polypeptide is selected from a group that includes XAF-1, XAF-2 N terminus XAF-2S, and XAF-2L. In other preferred embodiments, the XAF polypeptide is from a mammal (e.g., a human or a rodent), and the antibody is a polyclonal antibody, a monoclonal antibody, or a neutralizing antibody.
By xe2x80x9cXAFxe2x80x9d, xe2x80x9cXAF proteinxe2x80x9d, or xe2x80x9cXAF polypeptidexe2x80x9d is meant a polypeptide, or fragment thereof, which has at least 30%, more preferably at least 35%, and most preferably 40% amino acid identity to either the amino-terminal 131 amino acids of the human XAF-1 (SEQ ID NO.: 2) or the amino-terminal 135 amino acids of human XAF-2L (SEQ ID NO.: 10) polypeptides. It is understood that polypeptide products from splice variants of XAF gene sequences are also included in this definition. Preferably, the XAF protein is encoded by nucleic acid having a sequence which hybridizes to a nucleic acid sequences present in either SEQ ID NO.: 1 or SEQ ID NO.: 9 under stringent conditions. Even more preferably the encoded polypeptide also has XAF biological activity. Preferably, the XAF polypeptide has at least three zinc finger domains. More preferably, the XAF polypeptide has at least six zinc finger domains, at least five of which occur within 150 amino acids of the N-terminus.
By xe2x80x9czinc fingerxe2x80x9d is meant a binding domain capable of associating with zinc. A preferable zinc binding domain has the amino acid sequence 5xe2x80x2 Cxe2x80x94X2-5xe2x80x94Cxe2x80x94X11-18xe2x80x94C/Hxe2x80x94X2-5xe2x80x94C/H 3xe2x80x2 (SEQ ID NO.: 6), wherein xe2x80x9cXxe2x80x9d may be any amino acid. A more preferable zinc binding domain has the amino acid sequence 5xe2x80x2 Cxe2x80x94X1-2xe2x80x94Cxe2x80x94X11xe2x80x94Hxe2x80x94X3-5xe2x80x94C 3xe2x80x2 (SEQ ID NO.: 7), wherein xe2x80x9cXxe2x80x9d may be any amino acid. Even more preferably, a zinc binding domain has the amino acid sequence 5xe2x80x2 Cxe2x80x94X2xe2x80x94Hxe2x80x94X11xe2x80x94Hxe2x80x94X3xe2x80x94C 3xe2x80x2 (SEQ ID NO.: 8), wherein xe2x80x9cXxe2x80x9d may be any amino acid. Most preferably, a zinc binding domain is one found in a XAF polypeptide.
By xe2x80x9cXAF biological activityxe2x80x9d is meant any one or more of the biological activities described herein for XAF-1, XAF-2L, or XAF-2S, including, without limitation, the ability to bind an IAP (e.g., a XIAP), or another XAF polypeptide; the ability to cause apoptosis when transfected into a cell (particularly in a HeLa cell); the ability to enhance the NF-xcexaB inducing activity of a TRAF; and the ability to specifically bind a XAF-1, XAF-2L, or XAF-2S specific antibody.
By xe2x80x9cmodulating apoptosisxe2x80x9d or xe2x80x9caltering apoptosisxe2x80x9d is meant increasing or decreasing the number of cells that undergo apoptosis (than would otherwise be the case) in a given cell population. Preferably, the cell population is selected from a group including T cells, neuronal cells, fibroblasts, myocardial cells, or any other cell line known to undergo apoptosis in a laboratory setting (e.g., the baculovirus infected insect cells or an in vivo assay). It will be appreciated that the degree of modulation provided by a XAF polypeptide or a modulating compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change or a therapeutically effective change in the level of apoptosis which identifies a XAF polypeptide or a compound which modulates XAF or is a XAF therapeutic.
By xe2x80x9chigh stringency conditionsxe2x80x9d is meant hybridization in 2xc3x97SSC at 40xc2x0 C. with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see Ausubel, F. et al., 1994, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 6.3.1-6.3.6, hereby incorporated by reference.
By xe2x80x9cIAPxe2x80x9d is meant an amino acid sequence which has identity to baculovirus inhibitors of apoptosis. Mammalian IAPs include, without limitation, NAIP, HIAP1, HIAP2, and XIAP. Preferably, such a polypeptide has an amino acid sequence which is at least 45%, preferably 60%, and most preferably 85% or even 95% identical to at least one of the amino acid sequences of a baculovirus IAP.
By xe2x80x9cinhibiting apoptosisxe2x80x9d is meant any decrease in the number of cells which undergo apoptosis relative to an untreated control. Preferably, the decrease is at least 25%, more preferably the decrease is 50%, and most preferably the decrease is at least one-fold.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
By xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d means a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington""s Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is a XAF polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure XAF polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of a recombinant nucleic acid encoding a XAF polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides not only includes those derived from eukaryotic organisms but also those synthesized in E. coli or other prokaryotes. By xe2x80x9csubstantially pure DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cTRAFxe2x80x9d is meant a member of the TRAF family of proteins. TRAF family members each possess an amino terminal RING zinc finger and/or additional zinc fingers, a leucine zipper, and a unique, conserved carboxy terminal coiled coil motif, the TRAF-C domain, which defines the family. TRAF1 and TRAF2 were first identified as components of the TNF-R2 signaling complex (Rothe et al., Cell 78: 681-692, 1994). Preferred TRAF polypeptides are TRAF2, TRAF5, and TRAF6.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic mammals (e.g., rodents such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome.
By xe2x80x9cknockout mutationxe2x80x9d is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift mutation, or a missense mutation. Preferably, the mutation is an insertion or deletion, or is a frameshift mutation that creates a stop codon.
By xe2x80x9ctransformationxe2x80x9d is meant any method for introducing foreign molecules into a cell. Lipofection, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the teachings which may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organdies (e.g., and mitochondria and chloroplasts), bacteria, yeast, fungi, algae, animal tissue, and cultured cells.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a XAF polypeptide.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a XAF-1 polypeptide, a recombinant protein or a RNA molecule).
By xe2x80x9creporter genexe2x80x9d is meant any gene which encodes a product whose expression is detectable. A reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., luciferase or chloramphenicol acetyl transferase), toxicity (e.g., ricin), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labeled antibody).
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 or intron sequence regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By xe2x80x9cconserved regionxe2x80x9d is meant any stretch of six or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity between two or more of the XAF family members, (e.g., between human XAF-1 and another human XAF).
By xe2x80x9cdetectably-labeledxe2x80x9d is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32P or 35S) and nonradioactive labeling (e.g., chemiluminescent labeling, e.g., fluorescein labeling).
By xe2x80x9cantisense,xe2x80x9d as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, a XAF gene.
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., a XAF-1, XAF-2 N-terminus, XAF-2L, or XAF-2S specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds a XAF polypeptide but that does not substantially recognize and bind other non-XAF molecules in a sample, e.g., a biological sample, that naturally includes protein. A preferred antibody binds to the XAF-1 peptide sequence of FIG. 1 (SEQ ID NO.: 2). Another preferred antibody binds to the XAF-2 N-terminus peptide sequence of FIG. 35 (SEQ ID NO.: 4). Yet another preferred antibody binds to the XAF-2L peptide sequence of FIG. 37 (SEQ ID NO.: 10). Still another preferred antibody binds to the XAF-2S peptide sequence of FIG. 38C (SEQ ID NO.: 12). A more preferred antibody binds to two or more of XAF-1 (SEQ ID NO.: 2), XAF-2 N-terminus (SEQ ID NO.: 4), XAF-2L (SEQ ID NO.: 10) and XAF-2S (SEQ ID NO.: 12).
By xe2x80x9cneutralizing antibodiesxe2x80x9d is meant antibodies that interfere with any of the biological activities of a XAF polypeptide, particularly the ability of a XAF to participate in apoptosis. The neutralizing antibody may reduce the ability of a XAF polypeptide to participate in apoptosis by, preferably 50%, more preferably by 70%, and most preferably by 90% or more. Any standard assay of apoptosis, including those described herein, may be used to assess potentially neutralizing antibodies.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.